PROJECT SUMMARY While current Mycobacterium tuberculosis (Mtb) vaccine development has primarily focused on CD4+ and MHC Ia-restricted CD8+ T cell responses, increasing evidence shows that non-conventional CD8+ T cells restricted by MHC Ib molecules can recognize diverse microbial antigens and contribute to protection against Mtb infection. For example, H2-M3, Qa-1/HLA-E, Qa-2/HLA-G, MR1, and CD1 have been implicated in the host immune response against Mtb in mice and/or humans. However, most of these studies have only examined the function of MHC Ib-restricted T cells during the primary response to Mtb infection. Given that several subsets of MHC Ib- restricted T cells exhibit innate-like T cell characteristics and undergo unique thymic selection, it is unclear whether and which MHC Ib-restricted CD8+ T cell population can mount a memory response during Mtb infection. Furthermore, some MHC Ib molecules also act as regulators of the immune responses aside from their role in antigen presentation. In particular, Qa-1 can regulate immune responses against Mtb through the interaction with inhibitory CD94/NKG2A receptors and regulatory CD8+ T cells. To better understand the diverse role of MHC Ib molecules play in immune responses against Mtb, this proposal seeks to determine whether the composition and function of MHC Ib-restricted CD8+ T cell subsets in the secondary response differs from the primary response, as well as how individual Qa-1-mediated regulatory mechanisms contribute to the immune response against Mtb. In Aim 1, we propose to compare the kinetics, function, and protective ability of MHC Ib- restricted CD8+ T cell responses during primary and secondary Mtb infection in mice, and evaluate the protective efficacy of vaccination with Qa-1/HLA-E-restricted Mtb peptides. We will use a novel vaccination and re- challenge model that allows for complete clearance of an attenuated Mtb strain prior to challenge with fully virulent Mtb to study memory responses. These studies will allow us to identify the dominant MHC Ib-restricted CD8+ T cells and the Mtb antigens they present during the secondary immune response, their ability to mediate protective immunity against Mtb, and peptide vaccination strategies to increase the frequency of these effector CD8+ T cells. In Aim 2, we propose to investigate the mechanisms by which Qa-1 regulates immune responses during Mtb infection, with a focus on how inhibitory CD94/NKG2A receptors and suppressive Qa-1-restricted CD8+ Tregs individually contribute to the regulation of T cell responses during Mtb infection. To study these mechanisms, we will use mouse strains with genetic interruption of Qa-1?s interaction with individual receptors. These studies will allow us to identify how Qa-1 is able contribute to the balance between effector and regulatory immune responses for better protective immunity against Mtb. The combined results from our studies will yield a better understanding of how MHC Ib molecules and MHC Ib-restricted T cells contribute to the overall immune response against Mtb, for the development of better future vaccine strategies.